T cell therapy represents an attractive modality for the treatment of cancer due to its potential for high specificity, minimal toxicity and long-term immunoprotection. Advances in immunology, including the identification of T cell defined tumor- associated antigens, now enable the development of methods to augment an antigen-specific T cell response by the use of vaccines or adoptive T cell therapy. In contrast to vaccination strategies, the use of adoptively transferred T cells can overcome the in vivo constraints that influence the magnitude and avidity of the targeted response and provide for a uniform population of effector cells of defined specificity and phenotype. Our initial studies using adoptively transferred antigen-specific autologous CD8+ T cell clones for the treatment of patients with metastatic melanoma demonstrated that such effectors were safe, trafficked to tumor sites and mediated an antigen-specific immune response that resulted in favorable clinical outcomes. Limitations to efficacy were identified as shortened in vivo survival of CD8+ T cells and the outgrowth of antigen-loss variants. CD4+ T cells have been shown to provide a helper function to CD8+ T cells in vivo and may themselves mediate an anti-tumor effect through both direct and indirect means. Local activation of non-antigen-specific effectors by CD4+ T cells and the potentially synergistic CD4+ and CD8+T cell mediated anti-tumor response that may lead to antigen spreading in which broader multivalent responses are induced against non-targeted antigens will be investigated as possible mechanisms to prevent the outgrowth of antigen-loss variants. In addressing the limitations of the initial studies, the proposed project will evaluate in a series of early-phase clinical trials, the use of antigen-specific CD4+ and CD8+ T cells targeting the T cell-defined antigen, NY-ESO-1 for the treatment of patients with metastatic melanoma. The first trial will establish a safe dose and dosing schedule for administration of tumor-reactive, antigen-specific CD4+T cell clones. The second will evaluate the anti-tumor efficacy of CD4+ T cells alone at a dose and schedule established in the first trial. The third will evaluate the contribution of concurrently administered CD4+ T cells to the persistence, function and anti-tumor efficacy of adoptively transferred CD8+T cell clones. In the context of these clinical trials, the use of highly-defined T cell populations for adoptive transfer and precise immunologic assays for tracking and quantifying the numeric and functional persistence of the augmented targeted and non-targeted immune responses will provide insights to potential reasons for the success or failure of this strategy and facilitate an understanding of the requirements for tumor immunotherapy.